Investigating the community structure and diversity of arbuscular mycorrhizal (AM) fungi is important for accelerating the protection and application of mycodiversity, and for the sustainable development of ecosystems. Nevertheless, establishment of related research methods is the key to realizing and promoting such investigations. In this paper, we first provide a very brief historical survey on research methods of AM fungi, and introduce the principles and steps of both morphological and molecular biological methods, including spore morphological identification, polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and 454 pyrosequencing. Then, the results related to the long-term fertilization effects on soil AM fungal community structure and diversity revealed by spore calculation and identification, real-time PCR and PCR-DGGE, and 454 pyrosequencing were integrated for method comparison. Altogether, different methods could not only complement each other or have effects of mutual corroboration, but also have in consistencies and contradictions. Finally, the future development of research methods for AM fungi are proposed, including an upgrade of the pyrosequencing technique, and its combined application with biomarker methods, isotope labeling techniques, and others.